Abstract 14594: Dual Contrast-Enhanced Micro-Computed Tomography for Simultaneous Characterization of Macrocalcification and Tri-Layered Structural Remodeling in Explanted Human Aortic Valves

Abstract

Background: Micro-computed tomography (microCT) is excellent for detecting macrocalcification but ineffective at revealing other pathologically relevant structural details in the valves affected by aortic stenosis (AS). Here, we aimed to develop a dual contrast-enhanced microCT technique to simultaneously assess macrocalcification and layer-specific remodeling in diseased human aortic valves. Methods: Diseased (n=15) and normal (n=9) aortic valve leaflets were obtained from human subjects undergoing aortic valve replacement for AS and heart transplantation/autopsy, respectively. Leaflets were fixed (4% paraformaldehyde and 2% glutaraldehyde), stained with dual contrast (1% osmium tetroxide and 1% uranyl acetate), and embedded in epoxy resin. Additional unstained leaflets (n=3 diseased, n=3 normal), similarly processed otherwise, served as staining controls. MicroCT of embedded leaflets was performed using a ZEISS Xradia 520 Versa scanner, with low energy beam (80 kVp, 5 W, LE4 filter) and high-resolution acquisitions (15 μm x 15 μm, 5 sec exposure x 1601 projections). Reconstructed images were analyzed for leaflet/layer thicknesses at midline and macrocalcification volume using Dragonfly. Results: The tri-layered tissue structure was only visualized in stained but not unstained leaflets ( p =0.002). With staining, each layer was found to be thicker in the diseased than the normal leaflets (0.28±0.13 mm vs 0.18±0.07 mm, p =0.0002 for fibrosa; 0.64±0.68 mm vs 0.29±0.19 mm, p =0.012 for spongiosa; 0.32±0.16 mm vs 0.16±0.07 mm, p <0.0001 for ventricularis). The macrocalcification volume in the diseased leaflets was greater than that in the normal leaflets (84.7±84.4 mm 3 vs 0 mm 3 , p <0.0001) and correlated only with the thickness of spongiosa ( r =0.79, p <0.001) but not other layers ( p =0.67, p =0.26). Macrocalcification was as frequently observed in spongiosa as in fibrosa (66.7% vs 66.7% of diseased leaflets, p =0.3) but never in ventricularis. Conclusions: Dual contrast-enhanced microCT enables high-quality imaging of both macrocalcification and layer-specific remodeling in diseased human aortic valves. The results here suggest an intimate relationship between macrocalcification and spongiosa that warrants further investigation.